Exploring the potential of the model cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of various high-value terpenes

Background The robust model cyanobacterium Synechocystis PCC 6803 is increasingly explored for its potential to use solar energy, water and atmospheric CO2 for the carbon-neutral production of terpenes, the high-value chemicals that can be used for the production of drugs, flavors, fragrances and biofuels. However, as terpenes are chemically diverse, it is extremely difficult to predict whether Synechocystis is a suitable chassis for the photosynthetic production of various terpenes or only a few of them. Results We have performed the first-time engineering and comparative analysis of the best-studied cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of five chemically diverse high-value terpenes: two monoterpenes (C10H16) limonene (cyclic molecule) and pinene (bicyclic), and three sesquiterpenes (C15H24) bisabolene (cyclic), farnesene (linear) and santalene (cyclic). All terpene producers appeared to grow well and to be genetically stable, as shown by the absence of changes in their production levels during the 5–9-month periods of their sub-cultivation under photoautotrophic conditions). We also found that Synechocystis PCC 6803 can efficiently and stably produce farnesene and santalene, which had never been produced before by this model organism or any other cyanobacteria, respectively. Similar production levels were observed for cells growing on nitrate (the standard nitrogen source for cyanobacteria) or urea (cheaper than nitrate). Furthermore, higher levels of farnesene were produced by cloning the heterologous farnesene synthase gene in a RSF1010-derived replicating plasmid as compared to the well-used slr0168 neutral cloning site of the chromosome. Conclusions Altogether, the present results indicate that Synechocystis PCC 6803 is better suited to produce sesquiterpenes (particularly farnesene, the most highly produced terpene of this study) than monoterpenes (especially pinene). Supplementary Information The online version contains supplementary material available at 10.1186/s13068-022-02211-0.


Fig. S1B. Relevant part of the nucleotide sequence of the pCPS plasmid.
The NdeI (catATG) and EcoRI (gaattc) restriction sites were used for cloning the Pinus taeda α-pinene synthase gene downstream of the strong lambda-phage pR promoter (-35 box (TTGACT), -10 box (GATAAT) & transcription start site (A)) and associated ribosome binding site (AAGGAGG) of the pC vector.  (Table S1) was cloned as a NdeI-EcoRI restriction fragment in the pC plasmid, yielding pCSS that expresses the santalene synthase gene from the strong pR promoter (PR box). TT indicates the transcription terminators flanking the Sp R /Sm R marker.   (Table S1) was cloned as a NdeI-EcoRI restriction fragment in the pC vector, generating the pCFS plasmid that expresses the α-farnesene synthase gene from the strong pR promoter (PR box). TT stands for the transcription terminators flanking the Sp R /Sm R marker. a. Schematic representation of the relevant region of the pC (empty) plasmid vector and its derivative expressing the terpene synthase genes (large coloured arrows) from the strong lambda-phage pR promoter (small red trianges). PCR primers (pCF1_Fw and pCF1_Rv, additional file 1: Table S1B) and resulting DNA products are indicated by blue triangles and double arrows, respectively.

Restriction with NdeI and
b. typical UV-light image of the corresponding agarose gels used to analyze two clones of each studied strain excepted for pCSS. M: DNA size marker = 1 kb Plus DNA Ladder Thermo Scientific GeneRuler. C _ correspond to a negative control (no pC derived plasmid). a b Figure S1J. Construction of the Synechocystis strain harboring the Picea abies α-farnesene synthase gene in the slr0168 chromosome site. First, the pR-FS gene (expression of the α-farnesene synthase gene from the pR promoter) and the Km R marker were PCR amplified from the plasmids pCFS and pUC4K, respectively (Table S1). Second, the pTwist_NS-slr0168 plasmid was opened at its unique EcoRV restriction site flanked by the two 300 bp chromosomal DNA regions surrounding slr0168. Third, all three DNA cassettes (pR-FS, Km R and pTwist_NS-slr0168) were assembled by Gibson® cloning. The resulting pTwist_NS-slr0168-FS plasmid was transformed to Synechocystis to insert the pR-FS-Km R cassette in slr0168. Ion chromatograms (left panels) and corresponding mass spectra (right panels) of an α-pinene standard (upper panel) or dodecane samples (lower panel) of cultures of Synechocystis harboring either pC (negative control) or the pCPS (pinene production). S-(-)-limonene (retention time = 6.89 min) was used as the fixed-concentration (0.01 g.L -1 ) internal standard (IS) for quantification.

Synechocystis pCPS
Negative control Synechocystis pC

Fig. S3.C. GC-MS analyses of the dodecane overlay of cultures of the Synechocystis strains harboring the pC or pCLS plasmids.
Ion chromatograms (left panels) and corresponding mass spectra (right panels) of a S-(-)-limonene standard (upper panel) or dodecane samples (lower panel) of cultures of Synechocystis harboring either pC (negative control) or the pCLS (S-(-)-limonene production). Pinene (retention time 5.44 min) was used as the fixed-concentration (0.01 g.L -1 ) internal standard (IS) for quantification. The standard curve used to calculate the concentration of limonene was already published (Chenebault et al., 2020).

Synechocystis pCLS
Negative control Synechocystis pC Figure S3.D -Standard curve used to calculate the concentration of E-α-bisabolene in the dodecane overlay of cultures of the Synechocystis strain harboring the pCBS plasmid. Bisabolene (increasing concentrations) and trans-caryophyllene (fixed-concentration internal standard) were spiked in dodecane prior to GC-MS analysis. Due to the presence of several bisabolene isomers in the commercial standard, the contribution of (E)-α-bisabolene to the molarity was adjusted based on an attribution of 19.31  0.91% of total combined peak areas to the target molecules, as described (Wichmann et al., 2018).  c. d.

Synechocystis pCFS
Synechocystis pCBS Negative control Synechocystis pC

Fig. S3.F. -Standard curve used to calculate the concentration of α-farnesene in the dodecane overlay of cultures of the Synechocystis strains harboring the FS gene in either or both the plasmid (pCFS) and the chromosome (chrFS). Farnesene (increasing concentrations)
and trans-caryophyllene (fixed-concentration) were spiked in dodecane prior to GC-MS analysis. As the commercial standard contains several farnesene isomers, the contribution of α-farnesene to the molarity was adjusted based on an attribution of 7.78  0.37% of total combined peak areas to the target molecules, as described (Wichmann et al., 2018).  Figure S3.G. Standard curve used to calculate the concentration of -santalene in the dodecane overlay of cultures of the Synechocystis strain harboring the pCSS plasmid. -santalene (increasing concentrations) and nerolidol (fixed-concentration) were spiked in dodecane prior to GC-MS analysis. As the commercial standard contains several santalene, bergamotene, santalol and bergamotol isomers, the contribution of α-santalene to the molarity was adjusted based on an attribution of only 0.270.02% of total combined peak areas to the target molecules, as described (Wichmann et al., 2018).  Figure S3.H -Standard curve used to calculate the concentration of -exo-bergamotene in the dodecane overlay of cultures of the Synechocystis strain harboring pCSS. -exo-bergamotene (increasing concentrations) and nerolidol (fixed-concentration internal standard) were spiked in dodecane prior to GC-MS analysis. As the commercial standard contains several santalene, bergamotene, santalol and bergamotol isomers, the contribution of -exo-bergamotene to the molarity was adjusted based on an attribution of only 0.1% of total combined peak areas to the target molecules, as described (Wichmann et al., 2018).  Figure S3.I. -Standard curve used to calculate the concentration of epi-β-santalene in the dodecane overlay of cultures of the Synechocystis strain harboring pCSS. epi-β-santalene (increasing concentrations) and nerolidol (fixed-concentration internal standard) were spiked in dodecane prior to GC-MS analysis. As the commercial standard contains several santalene, bergamotene, santalol and bergamotol isomers, the contribution of epi-β-santalene to the molarity was adjusted based on an attribution of only 0.39%0.02% of total combined peak areas to the target molecules, as described (Wichmann et al., 2018).  Figure S3.J. GC-MS analyses of the dodecane overlays of cultures of the Synechocystis strain harboring the pCSS plasmid. m/z=93 ion chromatograms (upper panel) and mass spectra (lower panels) of the α-santalene (framed in mauve), epi-β-santalene (framed in purple) and α-exo-bergamotene (framed in pink) isomers were obtained from GC-MS analyses of a Synechocystis pCSS culture grown for 21 days. Nerolidol at 0.01 g.L -1 was used as the internal standard (IS) for quantification.